PB2168: P27: A NOVEL SUBSTRATE OF PDGFR
Authors: Carlos Miguel Castillo Giron, Heidelinde Jaekel, Ludger Hengst
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: p27Kip1 (CDK inhibitory protein 1) is a member of the cyclin-dependent kinase inhibitors (CKIs) of the Cip/Kip family. Is a typical tumour suppressor, however, it also possesses oncogenic properties. Deregulation of p27 leading to low protein levels or mislocalization are frequent in human malignancies and associated with poor prognosis. This protein level decline is mainly due to post-translational phosphorylation and ubiquitin-dependent degradation. p27 can be phosphorylated at multiple sites by kinases including FLT3. In 2008, the WHO classification of eosinophilic disorders included clonal/neoplastic eosinophilia resulting from Tyrosine Kinase (TK) fusion gene rearrangements as a new category, termed Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1. FIP1L1-PDGFRA fusion gene is the most common variant and it usually presents as chronic eosinophilic leukaemia. Aims: Being PDGFR - as FLT3- part of the Receptor Tyrosine Kinase (RTK) family, we hypothesize that PDGFR follows phosphorylation of p27, with the consequent ubiquitin-proteasome degradation, cell-cycle progression and ultimately cancer development. Methods: 1x106 293T cells were seeded in 10-cm the day before transfection. Transient transfection was performed using the calcium phosphate method. 24 hours after transfection, proteins were solubilized with 1% NP-40 containing lysis buffer, separated on 13% SDS–PAGE and analyzed by Western Blot. Bands were visualized by ECL- (GE-Healthcare). Immunoprecipitation was performed using antibodies covalently coupled to protein G-agarose. Results: 1)PDGFR𝛼 phosphorylates p27 on Tyrosine residues 74 and 88. To investigate the individual contribution of each of the three tyrosine residues present in p27 (Y74, Y88 and Y89), p27 proteins were expressed with one or several tyrosine residues mutated to phenylalanine together with PDGFR𝛼. Tyrosine phosphorylation of mutants lacking Y74 and Y88 was significantly reduced. Specific anti phospho-Tyrosine 74 and 88 antibodies were probed, demonstrating highly phosphorylation of p27 on residues Y74 and Y88 in vitro. 2)PDGFR𝛼 is in complex with p27. Pull down assay. HA-tagged PDGFR𝛼 and Flag-tagged p27. Flag-p27 co-precipitated PDGFR𝛼 demonstrating an in vitro stable interaction. 3)PDGFR𝛼 phosphorylation of p27 is Src-independent. Upon by-passing SRC signaling achieved by mutating PDGFR𝛼 on tyrosine residues 572/74 to phenylalanine, p27 remains phosphorylated by PDGFR𝛼, demonstrating a Src-independent phosphorylation. Summary/Conclusion: PDGFR𝛼 phosphorylates the CDK inhibitor p27 on Tyrosine residues 74 and 88. Myeloproliferative neoplasms resulting from fusion proteins like FIP1L1-PDGFR𝛼, its constitutive kinase activity may lead to p27 tyrosine phosphorylation Src-independent, with a consequent increased Skp2-dependent proteasomal degradation, converting the tumour suppressor ability to a potential oncogene.Keywords: PDGFRA, Cell cycle, Myeloproliferative disorder, FIP1L1-PDGFRA